Pectin is a major matrix polysaccharide found in the cell wall of plants. Pectins are composed of two distinct regions. The smooth region comprises of long stretches of homogalacturonan interrupted by rhamnose, and is relatively unbranched. The hairy region is rich in galacturonic and rhamnose residues and is highly branched. The side branches contain different sugars but primarily comprise the neutral sugar side chains, arabinan, .beta.-(1.fwdarw.4)-galactan and arabinogalactan. The function of these neutral sugar polysaccnaride side chains has not been fully established. It is speculated that they may function in modulating the pore size of the cell wall and therefore the mobility of proteins, possibly restricting access of various enzymes to their substrates. Moreover, the interaction of the side chains between themselves and with other cell wall polymers could contribute to the structure of the cell wall and the rheoiogical properties of products derived from them. In vitro studies carried out on a solution of apple pectin with different neutral sugar contents demonstrate that increase in branching of pectin results in higher zero-shear viscosity. It was concluded that this was due to pectin side chain interactions. In addition, more branched pectin gives higher elastic or storage moduli (G') than less branched pectin, suggesting that side chain of pectins contribute to elastic properties (Hwang et al., (1993) Food Hydrocolloids 7, 39-53).
The hydrolysis of .beta.-(1.fwdarw.4)-linked galactose from polymeric galactan side chains of pectin has been demonstrated in different plants and in various physiological states (de Vetten and Huber (1990) Physiol. Plant. 78, 447-454; Fischer and Bennett (1991) Ann. Rev. Plant Physiol Plant Mol. Biol. 42, 675-703). During the process of fruit ripening, the loss of the neutral sugar, galactose, is the single most extensive change in the cell walls of many fruits (Fischer and Bennett 1991). Galactose mobilization during fruit ripening has been demonstrated in several fruits including tomato, hot pepper, strawberry, apple, coffee, muskmelon, kiwi fruit, and nectarines. During the senescence of carnation petals, the decrease in cell wall yield is due largely (45%) to a loss of the neutral sugar galactose (de Vetten and Huber 1990). In germinating lupin cotyledons, up to 80% of galactose is mobilized primarily from the .beta.-(1.fwdarw.4)-linked galactan side chains of the rhamnogalacturonan backbone from secondary cell walls adapted to a storage function. A .beta.-galactosidase (exo-(1.fwdarw.4)-.beta.-D-galactanase) would be the enzyme activity predicted to be responsible for galactose mobilization from the galactan side chains of pectin.
.beta.-Galactosidase enzvme activities (including exo-galactanase activities) in plants have been described in the prior art (Dick et al., (1990) Physiol. Plant. 89, 369-375; Burns (1990) Phytochemistry 29, 2425-2429; Singh and Knox. (1985) Phytochemistry 24, 1639-1643; Kundu et al., (1980) Phytochemistry 29, 2079-2082). The purification of some .beta.-galactosidase enzymes has been described, though in many instances synthetic substrates rather than endogenous substrates have been used for enzvme characterization (Ogawa et al., (1990) Nippon Shokuin Kogyo Gakkaishi 37, 298-305; Giannakouros et al., (1991) Physiol. Plant. 82, 413-418). There is evidence that plant .beta.-galactosidases may be associated with developmental processes requiring cell wall turnover, like tissue elongation in Cicer arietinum epicotyl segments. In this tissue, .beta.-galactosidase has been demonstrated to he responsible for autolysis, and the natural substrate of the autolytic reaction is the pectic fraction of the cell wall (Dopico et al., (1989) Physiol. Plant. 75, 458-464; Valero and Labrador (1993) Physiol. Plant. 89, 199-203). A .beta.-galactosidase has been highly purified from the buffer-soluble fraction of carrot cell culture homogenate (Konno et al., (1986) Physiol. Plant. 68, 46-52). The enzyme was active on .beta.-(1.fwdarw.4)-linked galactan prepared from citrus pectin in an exo fashion. The loss of galactose in cell walls during softening has been widely documented (Bartley (1974) Phytochemistry 13, 2107-2111; Redgewell et al., (1992) Plant Physiol. 98, 71-81; Wegrzyn and MacRae (1992) Hort. Sci. 27, 900-902). In tomato, it has been found that the increase in monomeric galactose during fruit ripening is due to an increase in the rate of galactose solubilization from the cell wall rather than changes in the rate of metabolic utilization of the solubilized galactose (Kim et al., (1991) Postharvest Biol. Technol. 1, 67-80). This suggests the action of .beta.-galactosidases in vivo during fruit ripening. There have been several reports of increased .beta.-galactosidase activity during the process of fruit ripening (Bartley 1974; Pressey (1983) Plant Physiol. 71, 132-235; Ross et al., (1993) Planta 1889, 499-506). The .beta.-galactosidase purified from kiwifruit was active in cleaving terminal galactose attached at either the 2, 3, 4 or 6 position (Ross et al., 1993). In tomato fruits, Gross and Wallner (1979, Plant Physiol. 63, 117-120) have shown that decline in wall galactans precedes or accompanies increase in soluble polyuronide. Pressey (1983) has characterized three .beta.-galactosidase activities in ripening tomato fruits of which one (.beta.-galactosidase II), increases 3-fold during ripening. This enzyme was also able to degrade galactan extracted from the cell walls of green tomato and the author suggests a possible role for it in tomato softening. A .beta.-galactosidase has been purified from ripe coffee beans which increases four fold during the transition from immature to ripe fruits (Golden et al., (1993) Phytochemistry 34, 355-360). The enzyme displayed activity against galactan and arabinogalctan, however pectin yielded galactose only in conjunction with an endopolygalacturonase activity.
Solubilization of pectin during fruit ripening is a well-documented phenomenon (Fischer and Bennett, 1991). The action of endopolygalacturonase was considered to be the most likely cause of pectin solubilization, which was thought to be the cause of softening of fruits. Recent studies in transgenic tomato fruits argue against PG being the sole causal agent in the process of fruit softening (Giovannoni et al., (1989) Plant Cell 1, 53-63; Smith et al., (1990) Plant Mol. Biol. 14, 369-379). Recent evidence in fruits with no apparent endo or exo polyalacturonase activity suggests a role for .beta.-galactosidases active on the galactan side chains for the solubilization of pectin (Cutillas-Iturralde el al., (1993) Physiol. Plant. 89, 369-375). Ranawala et al., ((1992) Plant Physiol. 100, 1318-1325) have described a NaCl-released .beta.-galactosidase activity from cell walls of ripe muskmelon (Cucumis melo) fruits, that has the ability to degrade (in vitro) pectin extracted from pre-ripe fruits to smaller sizes of pectin, similar to those observed in ripe fruits. Moreover, there is no detectable PG activity at any stage of muskmelon fruit development and ripening. De Veau et al., (1993 Physiol. Plant. 87, 279-285) have demonstrated increased pectin solubility and decreased apparent molecular weight of pectin extracted from mature green tomato fruits when digested in vitro with .beta.-galactosidases isolated from avocado fruits. Though tomato pectin has been shown to contain at least 10% galactose, only 0.2% was mobilized using avocado derived .beta.-galactosidases. However, this minor change in pectin galactose composition was sufficient to change the solubility of polymeric pectin. These results suggest that an exo-galactanase might play an important role in the pectin solubilization during the process of fruit ripening.
For all the .beta.-galactosidase activities so far described, there are only some indications as to which of the macromolecular components of the cell walls are the actual in vivo substrates. An exception is the .beta.-galactosidase isolated from germinating nasturtium (Tropaeolum majus) cotyledons. The enzyme activity is coincident with xyloglucan mobilization, and the purified enzyme has the unique capability of hydrolysing the terminal .beta.-1,2-linked galactose from the galactoxylosyl sidechain of the xyloglucan polymer (Edwards et al., (1988) J. Biol. Chem. 263, 4333-4337). Buckeridge and Reid recently described (in the printed abstracts of disclosures made at the 6.sup.th Cell Wall Meeting, Nijmegen, Aug. 25-28, 1992, and at the Scottish Cell Wall Group Meeting, April 1993) the purification of a .beta.-galactosidase (an exo-(1.fwdarw.4)-.beta.-D-galactanase) that metabolises the linear .beta.-(1.fwdarw.4)-galactan component of the lupin cotyledonary cell wall. This enzyme is thought to play a key role in the post germinative mobilization of galactan. The enzyme activity is detectable only when galactan mobilization begins, increases during the period of galactan mobilization, and subsequently declines. The changes in exo-galactanase enzyme activity have been shown to correlate with changes in the level of the exo-galactanase enzyme, as determined by immunoblotting. This enzyme is highly specific to .beta.-(1.fwdarw.4)-galactan and does not hydrolyse other plant cell wall polysaccharides known to have terminal non-reducing galactose residues, like nasturtium xyloglucan (terminal (1.fwdarw.2)-.beta.-linked galactose) and larch arabinogalactan (terminal non-reducing (1.fwdarw.3) and (1.fwdarw.6) linked galactose residues.
The enzyme, exo-(1.fwdarw.4)-.beta.-D-galactanase, (which catalyses the hydrolysis of terminal galactose residues from (1.fwdarw.4)-.beta.-linked galactan side chains) appears to have an important role during several physiological processes. The present inventors have achieved the partial protein sequencing, cloning and sequence analysis of a full length cDNA coding for the exo-(1.fwdarw.4)-.beta.-D-galactanase from germinating agricultural lupins (Lupus angustifolius).